Neurological disease in xeroderma pigmentosum: prospective cohort study of its features and progression

Abstract Xeroderma pigmentosum (XP) results from biallelic mutations in any of eight genes involved in DNA repair systems, thus defining eight different genotypes (XPA, XPB, XPC, XPD, XPE, XPF, XPG and XP variant or XPV). In addition to cutaneous and ophthalmological features, some patients present with XP neurological disease. It is unknown whether the different neurological signs and their progression differ among groups. Therefore, we aim to characterize the XP neurological disease and its evolution in the heterogeneous UK XP cohort. Patients with XP were followed in the UK National XP Service, from 2009 to 2021. Age of onset for different events was recorded. Cerebellar ataxia and additional neurological signs and symptoms were rated with the Scale for the Assessment and Rating of Ataxia (SARA), the Inventory of Non-Ataxia Signs (INAS) and the Activities of Daily Living questionnaire (ADL). Patients’ mutations received scores based on their predicted effects. Data from available ancillary tests were collected. Ninety-three XP patients were recruited. Thirty-six (38.7%) reported neurological symptoms, especially in the XPA, XPD and XPG groups, with early-onset and late-onset forms, and typically appearing after cutaneous and ophthalmological symptoms. XPA, XPD and XPG patients showed higher SARA scores compared to XPC, XPE and XPV. SARA total scores significantly increased over time in XPD (0.91 points/year, 95% confidence interval: 0.61, 1.21) and XPA (0.63 points/year, 95% confidence interval: 0.38, 0.89). Hyporeflexia, hypopallesthaesia, upper motor neuron signs, chorea, dystonia, oculomotor signs and cognitive impairment were frequent findings in XPA, XPD and XPG. Cerebellar and global brain atrophy, axonal sensory and sensorimotor neuropathies, and sensorineural hearing loss were common findings in patients. Some XPC, XPE and XPV cases presented with abnormalities on examination and/or ancillary tests, suggesting underlying neurological involvement. More severe mutations were associated with a faster progression in SARA total score in XPA (0.40 points/year per 1-unit increase in severity score) and XPD (0.60 points/year per 1-unit increase), and in ADL total score in XPA (0.35 points/year per 1-unit increase). Symptomatic and asymptomatic forms of neurological disease are frequent in XP patients, and neurological symptoms can be an important cause of disability. Typically, the neurological disease will be preceded by cutaneous and ophthalmological features, and these should be actively searched in patients with idiopathic late-onset neurological syndromes. Scales assessing cerebellar function, especially walking and speech, and disability can show progression in some of the groups. Mutation severity can be used as a prognostic biomarker for stratification purposes in clinical trials.


Supplementary Text 1. Brief description of the Lasso method.
In the Lasso method, several regression models are estimated by increasing constraints on predictors' coefficients, i.e, progressively decreasing the coefficients towards the null value.
Such level of constraint is defined by a parameter designated as lambda (λ).For model selection, a cross-validation approach is used, fitting models iteratively with 4/5 of the data and testing their accuracy with the excluded 1/5.Two different criteria can be applied to select a model: a) optimal cross-validation criterium (λ of the model with the best crossvalidation fit); b) 1-standard error (1-SE) criterium (the model with a 1-SE more stringent level of constraint than the optimal λ value is chosen) (Supplementary Fig. 1).
Once a model is selected, two rules can be used to calculate model accuracy, i.e., the proportion of correct classifications: a) classification by the highest probability (data is assigned to the group with the highest probability); b) classification by the maximum odds ratio (data is assigned to the group with the highest odds ratio).Coefficients are presented as log(odds) for membership in each group.A threshold of log(odds)= |±0.4| approximately corresponds to OR= 1.5 for group membership, defining clinically significant effects.For the models considering SARA and ADL items, these variables were previously standardised to allow for comparisons in their effects.2 (9.5)  0 (0.0)  4 (18.2)  8 (47.1)  3 (42.9)  0 (0.0)  1 (12.5)  5 (41.7)  23 (24.7)Squamous cell carcinoma (SCC)

Supplementary Text 2. Description of the neuropathological findings in an XPD patient
who passed away at the age of 28.
By gross examination, there was generalised cortical atrophy with marked ventricular dilatation (Supplementary Fig. 5A-5C).The head of the caudate, the thalamus, the limbic structures and the brainstem were markedly atrophic (Supplementary Fig. 5B and 5C, Supplementary Fig. 6B and 6C).The cranial nerves also showed marked atrophy by naked eye.Mild atrophy was also seen in the cerebellum with a small dentate nucleus (Supplementary Fig. 6A).
Histology confirmed thinning of the cortical ribbon with loss of pyramidal neurons and increased glial cells in the frontal, parietal and temporal lobes (Supplementary Fig. 5D).The occipital lobe also showed marked neuronal loss, with relative preservation of the calcarine cortex (Supplementary Fig. 5E).The amygdala and the hippocampus displayed severe neuronal loss, with moderate thinning of the dentate fascia (Supplementary Fig. 5F and 5G).
All these areas showed loss of myelin in the white matter with gliosis.The corpus callosum was markedly thinned.The thalamus and the caudate nucleus showed marked neuronal loss, microvacuolation and gliosis (Supplementary Fig. 5H and 5I), whereas the globus pallidus, the putamen and the mammillary bodies were moderately affected.The claustrum was severely atrophic.There was prominent loss of pigmented neurons in the substantia nigra and locus coeruleus (Supplementary Fig. 6F).All brainstem nuclei, particularly the vestibulocochlear nuclei, showed severe neuronal loss (Supplementary Fig. 6G-6I).The Vth, VIIth and VIIIth cranial nerves were severely atrophic.In the Vth cranial nerve, myelin stain showed no obvious demyelination, with negative CD3 stain (for T-cells).CD68 revealed evenly distributed microglial cells and macrophages in the pons and the trigeminal nerve, respectively.Neurofilament stain (NF200KD) showed loss of axons.The overall features did not support acquired inflammatory demyelinating neuropathy.GFAP stain revealed mild reactive and prominent fibrillary astrocytosis in the brainstem.The cerebellum presented with marked Purkinje cells loss associated with Bergmann's gliosis and some axonal torpedoes, and thinning of the granular cell layer (Supplementary Fig. 6D and 6E).The cerebellar white matter showed some atrophy and myelin loss, and marked neuronal loss was present in the dentate nucleus.Extensive immunohistochemistry was performed and showed negative results with -amyloid, hyperphosphorylated tau, TDP43, p62 and -synuclein, excluding all common neurodegenerative disorders.a Data is presented as count, and the percentage over the total number of patients with the abnormal test in each complementation group, unless otherwise stated.

Figure 1 .
Graphical representation of the criteria used for model selection in Lasso methods.The y-axis represents model multinomial deviance, that is, a measure of model goodness-of-fit.The top x-axis represents the number of predictors included in the model.The bottom x-axis represents the level of model constraint (λ value).(A) Model chosen with optimal cross-validation criterium.(B) Model chosen with 1-SE criterium.Image courtesy of Prof Douglas Langbehn.

a
Values represent number of subjects with percentages relative to the group size.Supplementary Figure 2. Age of onset for skin symptoms in the different complementation groups.(A) Kaplan-Meier (KM) plot showing survival curves for each one of the groups (global log-rank test, χ 2 = 65.1, df= 5, P< 0.001).(B) Dot plot representing individual values for age of onset of cutaneous symptoms in those subjects who presented the event of interest.

Supplementary Figure 4 .
Age of occurrence of the first cutaneous neoplasm in the different complementation groups.(A) Kaplan-Meier (KM) plot showing survival curves for each one of the groups (global log-rank test, χ 2 = 28.1,df= 5, P< 0.001).(B) Dot plot representing individual values for age of onset of cutaneous neoplasms in those subjects who presented the event of interest.

Figure 5 .
Neuropathological findings in brain tissue of a patient diagnosed with xeroderma pigmentosum complementation group D (XPD), aged 28.(A) Marked generalised cortical atrophy with thickened leptomeninges.(B)-(C) Coronal sectioning of the left half brain showing deep sulci, markedly dilated ventricles, and prominent atrophy of the caudate nucleus and the thalamus.The amygdala and the hippocampus are also small (C).(D) Severe loss of neurones, particularly pyramidal cells, in the frontal cortex.(E) The calcarine cortex shows no obvious neuronal loss.(F)-(G) The hippocampus reveals subtotal neuronal loss, and the dentate fascia is also thinned.(H)-(I) The thalamus shows atrophy and marked neuronal loss in higher magnification (I).(D), (E), (G) and (I): haematoxylin-eosin.(F) and (H): Luxol fast blue-Nissl stain.Scale bars: Mild cerebellar cortical atrophy with small dentate nucleus.(B) Depigmentation of the substantia nigra in the midbrain.(C) Marked atrophy of the pons and the medulla oblongata.The cranial nerves are also very thin.(D) Severe loss of Purkinje cells and thinning of the granular cell layer.(E) Axonal torpedo (arrow) in the granular cell layer below a rare surviving Purkinje cell.(F) Hardly any surviving neurons in the substantia nigra.(G)-(H) Marked atrophy of the medulla oblongata.(I) Severe neuronal loss in the lateral vestibular nuclei by higher magnification.(D)-(G), (I): haematoxylin-eosin.(H): Luxol fast blue-Nissl stain.Scale bars: (D): 150m; (E), (F) and (I): 80m; (G) and (H): 1mm.

aFindings
Data is presented as count, and the percentage over the total number of patients with the abnormal test in each complementation group, unless otherwise stated.EMG/NCS: electromyogram/nerve conduction studies.N/A: not applicable.

Supplementary Table 2 Type of first symptom at onset for the different complementation groups a
a Variables are expressed as number of subjects with percentages relative to each group size.Others include: photophobia, conjunctival injection, hypopigmented macules, presymptomatic stage or combination of several symptoms.

Supplementary Figure 3. Age of onset for eye symptoms in the different complementation groups. (A) Kaplan
-Meier (KM) plot showing survival curves for each one of the groups (global log-rank test, χ 2 = 21.0,df= 5, P< 0.001).(B) Dot plot representing individual values for age of onset of ocular symptoms in those subjects who presented the event of interest.

Supplementary Table 8 Estimated SARA total mean progression rates as a function of follow-up time, time-since-onset of the first disease manifestation, and time-since-onset of the first neurological symptoms a Complementation group Progression rate per year of follow-up time (unadjusted) Progression rate per year of follow-up time (adjusted) Progression rate per year since onset (first disease symptom)
Results represent estimated mean progression rates for SARA total score in each complementation group (accounting for intrasubject variability), with their 95% CIs.The first two models represent the estimated mean progression rates per year of observed follow-up time (unadjusted and adjusted by baseline age, respectively).The third model corresponds to the estimates per year since onset of the first symptoms of the condition.The fourth model shows the estimates per year since onset of neurological symptoms.This last model only considered those subjects with self-reported onset of neurological manifestations and, therefore, estimates were not calculated in XPC, XPE and XPV, as most subjects in these groups did not refer an onset of neurological manifestations.
a N/A: not applicable.

Supplementary Table 9 Longitudinal analysis of SARA items a
a Results represent estimated mean progression rates (points per year), as a function of follow-up time, for each score in each complementation group (accounting for intrasubject variability), with their 95% CIs.χ 2 values represent the test for global differences among progression rates in the groups.If such test is significant (P< 0.05), pairwise contrasts are calculated and corrected for multiple comparisons via the Tukey's method.Statistically significant contrasts for each item (P< 0.05) are summarised in the last column.

Supplementary Table 10 Cross-sectional analysis of ADL items with repeated measures ANOVA with random groups effects a
Results represent estimated mean scores in each complementation group (accounting for intrasubject variability), with their 95% CIs.χ 2 values represent the test for global differences among the groups.If such test is significant (P< 0.05), pairwise contrasts are calculated and corrected for multiple comparisons via the Tukey's method.Statistically significant contrasts for each item (P< 0.05) are summarised in the last column.Results represent estimated mean progression rates (points per year), as a function of follow-up time, for each score in each complementation group (accounting for intrasubject variability), with their 95% CIs.χ 2 values represent the test for global differences in progression rates among the groups.If such test is significant (P< 0.05), pairwise contrasts are calculated and corrected for multiple comparisons via the Tukey's method.Statistically significant contrasts for each item (P< 0.05) are summarised in the last column.
a a

Table 12 Estimated mean frequencies for all INAS items in the different complementation groups a
a Frequencies are presented as percentages.Frequencies were calculated using Bayesian methods to account for intrasubject variability.LL: lower limb.UL: upper limb.